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(Circulation Research. 1995;77:1070-1076.)
© 1995 American Heart Association, Inc.

Articles

Multiple Growth Factors Modulate mRNA Expression of Angiotensin II Type-2 Receptor in R3T3 Cells

Toshihiro Ichiki, Yoshikazu Kambayashi, Tadashi Inagami

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn.

Correspondence to Tadashi Inagami, PhD, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

	Abstract

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Abstract Previous studies showed that angiotensin II type-2 receptor (AT₂) sites were increased when R3T3 cells were growth arrested and decreased when they were stimulated with fibroblast growth factor or serum. We examined the effects of several other growth factors on the expression of AT₂ mRNA to clarify the relation between the AT₂ receptor and growth factors. R3T3 cells were cultured in the medium containing 10% FCS until they were confluent, and then serum was removed. AT₂ mRNA was increased after serum was depleted, and the expression level reached a plateau after 2 days of serum depletion. The presence of serum (10%), fibroblast growth factor (10 ng/mL), or lysophosphatidic acid (1 µmol/L) reduced the AT₂ mRNA expression. Phorbol ester (1 to 100 nmol/L) also suppressed the AT₂ mRNA expression in a dose-dependent manner. Interleukin-1ß (1 ng/mL) enhanced the AT₂ mRNA expression 1.6-fold and the AT₂ receptor number 1.4-fold. Insulin (100 nmol/L) enhanced AT₂ mRNA expression 1.4-fold and the AT₂ receptor number 1.6-fold. These results suggest that AT₂ mRNA expression is modulated by multiple growth factors in both positive and negative directions. The presence of potential cis DNA elements that respond to interleukin-1ß (CCAAT enhancer binding protein site), insulin [insulin response sequence of phospho(enol)pyruvate carboxykinase gene], and phorbol ester (AP-1 site) in the promoter region of the mouse AT₂ gene suggests that the effects of these growth factors and phorbol ester may be mediated via these cis DNA elements.

Key Words: AT₂ receptors • mRNA expression • R3T3 cells • growth factor

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Angiotensin (Ang) II plays an important role in blood pressure regulation, fluid homeostasis, and drinking behavior. In addition, a growing body of evidence suggests that Ang II is a growth factor.^{1 2} Biological effects of Ang II on target cells such as the adrenal gland, vascular smooth muscle cells, and neurons are mediated by Ang II receptors. Two isoforms of Ang II receptor have been known and cloned. Although both receptors have putative seven-transmembrane domains, amino acid sequence homology is very low.^{3 4 5 6 7 8 9}

Almost all known biological effects traditionally ascribed to Ang II, such as vasoconstriction, aldosterone release, water drinking, cell proliferation, and facilitation of adrenergic flow, are mediated by the AT₁ receptor, which is coupled positively to phospholipase C and Ca²⁺ channel and negatively to adenylyl cyclase.^{1 2}

As for the AT₂ receptor, its signaling mechanism and coupling to G protein are controversial.^{10 11 12 13 14 15} Emerging evidence, however, suggests that stimulation of the AT₂ receptor inhibits cell growth¹⁶ and that the AT₂ receptor mediates some hemodynamic effects, such as vasodilatation,¹⁷ free water clearance,¹⁸ and cerebrovascular resistance.¹⁹

The AT₂ receptor shows unique tissue-specific and ontogeny-dependent expression.^{20 21 22} The AT₂ receptor is expressed highly in fetal tissues, most conspicuously in mesenchymal tissues²⁰ and specific brain nuclei of rats.²² This expression decreases rapidly after birth.^{20 22} In adult rats, the AT₂ receptor is expressed in the adrenal medulla,²³ heart,^{24 25} several brain nuclei,²² and myometrium.²⁶ These studies suggest the developmental, reproductive, and neuronal roles of Ang II via the AT₂ receptor on the basis of spatiotemporally regulated expression of the AT₂ receptor.

In vitro, R3T3 cells,^{27 28} a mouse fibroblast cell line, and PC12W cells,^{10 29} a rat pheochromocytoma cell line, were reported to express AT₂ receptors. In R3T3 cells, AT₂ receptor sites were decreased by basic fibroblast growth factor (bFGF) and bombesin.²⁸ The AT₂ sites in PC12W cells were decreased by incubation with nerve growth factor.²⁹ To the best of our knowledge, no growth factors or chemicals were reported to enhance the AT₂ receptor expression in these cells except for homologous upregulation of AT₂ sites by Ang II.²⁸

Recently, we have cloned and sequenced the promoter region of the mouse AT₂ receptor gene.³⁰ There are several potential cis DNA elements, such as AP-1, C/EBP, and an IRS of the PEPCK gene promoter. The AP-1 site is bound by a Fos and Jun heterodimeric transcription factor^{31 32} when stimulated by growth factors such as PDGF and FGF. This effect is mimicked by phorbol ester, which activates PKC and Fos/Jun. The C/EBP site is occupied by a homodimeric or heterodimeric complex of the C/EBP transcription factor family upon stimulation by IL-1ß.^{33 34}

Previous studies have shown that growth-arresting conditions enhance the expression of the AT₂ receptor sites and growth factors suppress it.^{28 29 35} We have examined the effect on the expression of AT₂ mRNA of several other growth factors, which were chosen on the basis of the potential hormone or growth factor–responsive elements present in the promoter region of the mouse AT₂ gene. We have studied the effect of IL-1ß, insulin, FGF, TPA, and LPA on the expression of AT₂ mRNA in R3T3 cells.

	Materials and Methods

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Reagent
DMEM, FCS, penicillin, and streptomycin were purchased from GIBCO BRL. Bovine FGF, porcine insulin, BSA, TPA, and LPA were purchased from Sigma Chemical Co. IL-1ß was from Upstate Biotechnology Inc. [¹²⁵I]KI and [³²P]-dCTP were purchased from Du-Pont NEN.

Cell Culture
R3T3 cells were maintained in the DMEM supplemented with 10% FCS, 1000 U/mL penicillin, and 1000 µg/mL streptomycin. Cells were cultured under 5% CO₂ and at 37°C. Cells (1x10⁶) were seeded in 10-cm tissue culture dishes and cultured in the presence of serum for 3 days. At this point cells were almost confluent, and medium was changed to DMEM supplemented with 0.2% BSA (referred to as serum-free medium in the text) and cultured for additional days as indicated in "Results."

Northern Blot Analysis
Total RNA was prepared from R3T3 cells by the acid guanidium–phenol–chloroform extraction method.³⁶ Fifteen micrograms of total RNA was electrophoresed in a 1.0% agarose/1.0% formaldehyde gel and transferred to Hybond N⁺ membrane (Amersham) by a capillary transfer method in 10x SSC (1x SSC is 300 mmol/L NaCl and 30 mmol/L sodium citrate) buffer overnight. The membrane was baked for 2 hours at 80°C. Prehybridization and hybridization were performed in a buffer containing 50% formamide, 5x SSC, 80 mmol/L sodium phosphate (pH 7.5), 2x Denhardt's solution, 1% SDS, and 100 ng/mL heat-denatured herring sperm DNA for 2 hours and 16 hours, respectively, at 42°C. A full-length cDNA of mouse AT₂ gene and the coding region of , ß, and C/EBP cDNA were labeled with ³²P by a Prime It kit (Stratagene) and used as a probe after heat denaturation. The hybridized filter was washed twice with 2x SSC for 5 minutes at room temperature, followed by two washes with 2x SSC/1% SDS for 30 minutes at 55°C. The filter was then exposed to Kodak X-OMAT film at -70°C. The hybridized filter was stripped by boiling in 0.5% SDS solution and hybridized to a ³²P-labeled ß-actin probe to obtain reference for the amount of applied RNA. Autoradiographic analysis was performed by an image scanner (ES-800C Scanner, Epson America Inc) and a computer program (NIH IMAGE 1.49). After scanning the autoradiogram, an appropriate window to determine the density of the band was set. Specific density was determined by subtracting the density of the blank lane from that of samples.

Scatchard Plot Analysis
¹²⁵I-labeled Sarile was prepared by the conventional lactoperoxidase method. The number of AT₂ binding sites was estimated by the displacement of binding of ¹²⁵I-Sarile by PD123319, an AT₂ receptor–specific antagonist. Confluent R3T3 cells in 24-well dishes were cultured with or without growth factors or phorbol ester for 48 hours, washed twice with HBSS, and incubated for 120 minutes at room temperature with various concentrations of ¹²⁵I-Sarile in DMEM supplemented with 0.2% BSA. Then cells were washed three times with ice-cold HBSS and solubilized in 0.5 mL of 0.2N NaOH. An aliquot (0.2 mL) was subjected to counting radioactivity, and 0.1 mL was subjected to the quantification of protein content.

Statistics
Statistical analyses of the relative AT₂ mRNA expression, dissociation constant (K_d), and AT₂ receptor sites (B_max) were performed by ANOVA and post hoc Duncan's test if appropriate. Values of P<.05 were considered statistically significant.

	Results

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AT₂ receptor sites were reported to increase after cells were confluent, especially when they were cultured in serum-free medium.²⁸ R3T3 cells (1x10⁶ cells in a 10-cm tissue culture dish) were cultured in medium containing 10% FCS for 3 days, when cells were almost confluent (Fig 1, lane 1), and then serum was removed and cultured in serum-free medium for additional days (Fig 1, lanes 2 to 4). Total RNA was prepared from these cells and examined by Northern blot analysis. mRNA of AT₂ was increased until 2 days of serum depletion; however, no difference was observed between the expression level of the AT₂ mRNA after 2 and 4 days of serum depletion.

View larger version (31K): [in this window] [in a new window]   Figure 1. Effect of serum depletion on AT2 mRNA expression in R3T3 cells. R3T3 (1x106) cells in 10-cm tissue culture dishes were cultured in medium containing 10% FCS for 3 days, when they were almost confluent (lane 1). Then serum was depleted and cultured for additional days, as indicated on the top of the figure (lanes 2 through 4). The total RNA was extracted from each sample, and 15 µg total RNA was electrophoresed in 1% agarose/1% formaldehyde gel, transferred to a nylon membrane, and hybridized with 32P-labeled mouse AT2 cDNA. The figure is representative of three independent experiments.

Therefore, in all of the subsequent experiments, the expression level of the AT₂ mRNA in R3T3 cells was examined after 2 days of serum depletion or 2 days of stimulation by growth factors in serum-free medium.

Effect of Serum, FGF, TPA, and LPA on the Expression of AT₂ mRNA
We examined the effect of serum and FGF on the expression of AT₂ mRNA. Previously, AT₂ receptor sites were reported to decrease after exposure to these stimuli.²⁸ As shown in Fig 2, 10% FCS and 10 ng/mL FGF suppressed the AT₂ mRNA expression. At this concentration, FGF was more potent in suppressing the AT₂ mRNA expression than was 10% serum.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of bFGF and serum on the expression of AT2 mRNA. R3T3 cells were cultured in serum-containing medium for 3 days and then cultured for an additional 2 days in serum-free medium (lane 1), serum-free medium with 10 ng/mL bFGF (lane 2), or medium with 10% FCS (lane 3). Fifteen micrograms of the total RNA from each sample was examined by Northern blot analysis, as in Fig 1. The figure is representative of three independent experiments.

Because cellular responses to LPA have been reported to show a striking similarity with responses to serum,³⁷ we examined the effect of LPA on the expression of AT₂ mRNA. LPA dose-dependently suppressed AT₂ mRNA (Fig 3).

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of LPA on the expression of AT2 mRNA. R3T3 cells were cultured in serum-containing medium for 3 days, and then the medium was changed to serum-free medium and cultured for an additional 2 days (lane 1). LPA was added to various concentrations, as indicated at the top of the figure, during culture in serum-free medium (lanes 2 through 4). Fifteen micrograms of the total RNA from each sample was examined by Northern blot analysis, as in Fig 1. The figure is representative of three independent experiments.

We have previously reported that PDGF suppressed the AT₂ receptor sites in vascular smooth muscle cells.³⁵ Both PDGF and FGF receptors are coupled to phospholipase C-,^{38 39} resulting in the activation of PKC. There is an AP-1 site that may respond to the activation of PKC by growth factors or TPA in the promoter region of the mouse AT₂ gene. We examined the effect of TPA on the expression of AT₂ mRNA. Fig 4 shows that TPA suppressed the AT₂ mRNA expression in a dose-dependent manner. TPA as low as 1 ng/mL (lane 2) was effective in suppressing AT₂ mRNA expression.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of TPA on the expression of AT2 mRNA. R3T3 cells were cultured in serum-containing medium for 3 days, and then the medium was changed to serum-free medium and cultured for an additional 2 days (lane 1). TPA was added to various concentrations, as indicated at the top of the figure, during culture in serum-free medium (lanes 2 through 4). Fifteen micrograms of the total RNA from each sample was examined by Northern blot analysis, as in Fig 1. The figure is representative of three independent experiments.

Effect of Insulin on the Expression of AT₂ mRNA
Since the 11-nucleotide sequence found in the promoter region of the mouse AT₂ gene has some homology with the IRS present in the promoter region of the PEPCK gene,⁴⁰ we examined the effect of insulin on the expression of the AT₂ mRNA. Fig 5 shows that 100 nmol/L insulin enhanced the AT₂ mRNA expression 1.4-fold. Insulin at 1 µmol/L also enhanced AT₂ mRNA expression 1.2-fold. Scatchard plot analysis showed that B_max was increased 1.6-fold, whereas K_d was unchanged (Table) by stimulation with 100 nmol/L insulin.

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of insulin on the expression of AT2 mRNA. R3T3 cells were cultured in serum-containing medium for 3 days, and then the medium was changed to serum-free medium and cultured for an additional 2 days (lane 1). Insulin was added to various concentrations, as indicated at the top of the figure, during culture in serum-free medium (lanes 2 through 4). Fifteen micrograms of the total RNA from each sample was examined by Northern blot analysis, as in Fig 1. The figure is representative of three independent experiments.

View this table: [in this window] [in a new window]   Table 1. Kd and AT2 Receptor Sites in Growth Factor–Stimulated R3T3 Cells

Effect of IL-1ß on the Expression of AT₂ mRNA
IL-1ß is known to activate gene transcription by activating C/EBP transcription factors, which bind to the C/EBP site of the promoter region. Because there is a C/EBP-like sequence in the promoter region of the mouse AT₂ gene, we examined the effect of IL-1ß on the expression of the AT₂ mRNA. IL-1ß (1 ng/mL) enhanced the AT₂ mRNA expression 1.6-fold (Fig 6). Scatchard plot analysis showed that AT₂ receptor sites in IL-1ß–stimulated R3T3 cells were increased 1.4-fold (Table). We further examined the effect of IL-1ß on the mRNA expression of three C/EBP isoforms in R3T3 cells. Expression of mRNA in the ß and isoforms of C/EBP was enhanced by IL-1ß stimulation (data not shown), whereas IL-1ß showed no effect on the expression level of mRNA in the isoform of C/EBP.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of IL-1ß on the expression of AT2 mRNA. R3T3 cells were cultured in serum-containing medium for 3 days, and then the medium was changed to serum-free medium and cultured for an additional 2 days (lane 1). IL-1ß was added to various concentrations, as indicated at the top of the figure, during culture in serum-free medium (lanes 2 and 3). Fifteen micrograms of the total RNA from each sample was examined by Northern blot analysis, as in Fig 1. The figure is representative of three independent experiments.

Effects of these growth factors or TPA on the expression of the AT₂ mRNA is summarized in Fig 7. The autoradiogram was scanned and analyzed by a computer. The density of the AT₂ mRNA was normalized by the density of actin mRNA. The expression level of the AT₂ mRNA in R3T3 cells cultured in serum-free medium for 2 days after cells were confluent was referred to as control and set to 100%. bFGF, serum, TPA, and LPA significantly suppressed the relative AT₂ mRNA expression, and insulin and IL-1ß significantly enhanced it.

View larger version (40K): [in this window] [in a new window]   Figure 7. Relative AT2 mRNA expression in unstimulated and growth factor–stimulated R3T3 cells. Autoradiographic analysis of the Northern blot was performed by scanning and computer program (NIH IMAGE 1.49). The density of the AT2 mRNA was normalized in reference to the density of ß-actin mRNA. The expression level of AT2 mRNA in R3T3 cells cultured in serum-free medium for 2 days after cells were confluent was referred to as control and set to 100%. bFGF, serum, and TPA significantly suppressed the relative AT2 mRNA expression (*P<.05), and insulin and IL-1ß significantly enhanced it (**P<.05).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The AT₂ receptor is abundantly expressed in fetal tissues, particularly mesenchymal tissues²⁰ and some brain nuclei.^{21 22} This AT₂ receptor expression is rapidly shut off or decreased after birth.^{20 22} In adult rats, the AT₂ receptor is expressed in some brain nuclei,²² heart,^{24 25} myometrium,²⁶ and adrenal medulla.²³ Adult human lung, however, was reported to continue to express AT₂ mRNA.⁴¹ The differential expression pattern of the AT₂ receptor between the fetus and adult rat suggests that a differential control mechanism of gene transcription is adopted during its ontogeny.

R3T3 cells are derived from Swiss 3T3 mouse embryonic fibroblasts and exclusively express the AT₂ subtype of the Ang II receptor.²⁸ So far, other subclones of 3T3 cells have not been reported to express AT₂.²⁸ Most of the cells that abundantly express AT₂ in fetal mesenchymal tissue are undifferentiated fibroblasts.²⁰ The identical origin and abundant expression of AT₂ in these cells suggest that regulation of AT₂ gene expression in R3T3 cells represents that of fetal mesenchymal tissues. Therefore, R3T3 cells may be a good model for the study of the transcriptional control of the AT₂ gene, and the expression of the AT₂ gene in this cell line may reflect the transcriptional regulation of the AT₂ gene in the fetus. The AT₂ mRNA was increased after cells reached confluence and serum was removed. These data were in good agreement with a previous binding study using radiolabeled ligand.²⁸ Since it was observed that rapidly growing R3T3 cells express the AT₂ mRNA at a very low level whereas the expression level was markedly increased after cells were confluent and serum was removed, it is suggested that the cell cycle may control the transcription of the AT₂ gene. Whether the quiescent state activates the transcription of the AT2 gene or the actively growing state inhibits the transcription is not clear.

Recently, it has been reported that the effects of serum are traced to LPA.³⁷ Both serum and LPA suppressed AT₂ mRNA and AT₂ sites. LPA seems an important factor in serum that suppresses the AT₂ gene expression. However, it is not clear whether all the suppressive effects of serum can be ascribed to LPA.

Peptide growth factors such as bFGF²⁸ and PDGF³⁵ were reported to suppress the AT₂ receptor number. Therefore, changes of humoral environment, such as elevated growth factor concentration, may provide an explanation for a rapid decrease in the AT₂ receptor number after birth in mesenchymal tissue and certain brain nuclei. FGF and PDGF receptors were shown to couple to phospholipase C-, which results in the activation of PKC as well as Fos and Jun transcription factors, which in turn bind to the AP-1 site of the promoter region of many genes. This effect is mimicked by phorbol ester. Because both bFGF and TPA suppressed the AT₂ mRNA expression, their effects may converge on the AP-1 site. The concentration of TPA used in the present study is very low. Suppression of the AT₂ mRNA was observed at as low as 1 ng/mL TPA. Therefore, the effect of TPA on the expression of AT₂ mRNA is probably due to activation rather than downregulation of PKC, since TPA at such a low concentration does not downregulate PKC.

IL-1ß upregulates gene transcription by enhancing the expression of the C/EBP transcription factor. C/EBP belongs to the family of the basic leucine zipper–type transcription factor, which forms a homodimeric or heterodimeric complex. To date, five distinct members of C/EBP (, ß, , and C/EBP and CHOP 10) have been reported.^{42 43 44 45 46 47 48 49 50} The expression of mRNA of three species whose cDNAs were available was examined by Northern blot analysis. In R3T3 cells, IL-1ß enhanced the expression level of the ß and C/EBP mRNA (data not shown), whereas C/EBP mRNA was constitutively expressed and was not affected by IL-1ß in R3T3 cells. Therefore, ß and C/EBP transcription factors could be candidates responsible for the upregulation of the AT₂ mRNA. Alternatively, a heterodimeric complex of ß or C/EBP with C/EBP may be functionally important. The effect of IL-1ß on the expression of C/EBP and CHOP 10 in R3T3 cells remains to be determined. AT₂ has been reported to be expressed within the superficial dermis of the skin surrounding the wound.⁵¹ IL-1ß is one of the important cytokines mediating inflammation.⁵² It is possible that IL-1ß plays a role in AT₂ induction during the process of inflammation (eg, as in wound healing).

Although not all genes that respond to insulin stimulation have a common responsive sequence in their promoter region, the 11-nucleotide sequence of the promoter region of the mouse AT₂ gene is very similar to the IRS; both sequences are commonly found in the PEPCK, -amylase, and gene-33 promoter regions.⁴⁰ mRNA expression of these genes is reported to be enhanced or suppressed by insulin. Therefore, the 11-nucleotide sequence in the promoter region of the mouse AT₂ gene may be a candidate responsible for the upregulation of the AT₂ mRNA by insulin. The physiological role of insulin for the upregulation of AT₂ is not clear. Fetal mesenchymal fibroblasts, which highly express AT₂, are reported to express a substantial amount of insulin-like growth factor-I and its receptor.⁵³ Insulin may be important for the abundant expression of AT₂ in fetal mesenchymal tissues.

Fig 8 shows relative positions of the potential cis DNA elements in the promoter region of the mouse AT₂ gene, and their nucleotide sequences are compared with consensus cis DNA elements of the IRS of the PEPCK gene, C/EBP, and AP-1.

View larger version (16K): [in this window] [in a new window]   Figure 8. Relative positions of potential cis DNA elements present in the promoter region of the mouse AT2 gene. The restriction enzyme sites and relative locations of the potential cis DNA elements are indicated. Abbreviations of restriction enzymes are as follows: B, BamHI; E, EcoRI; and S, Sac I. The white box indicates the first exon and arrow indicates transcription initiation sites of the mouse AT2 gene. The nucleotide sequences of the potential cis DNA elements present in the promoter region of the mouse AT2 gene are compared with the consensus sequence of the transcriptional element of IRS of the PEPCK gene, C/EBP and AP-1. N indicates any nucleotide.

The steady state mRNA level represents the balance of transcription and degradation. We have not determined whether these growth factors exert their effects at the transcriptional level or mRNA degradation. Further studies on the molecular mechanism of the AT₂ mRNA expression, particularly the effect of growth factors on gene transcription and mRNA stability, are needed.

Although earlier studies^{28 29 37} showed that growth-arresting conditions enhance the expression of AT₂ receptor sites and that stimulation by growth factors suppressed it, we have found another group of growth factors that enhance the expression of AT₂ receptor sites. These results shed new light on the growth factor regulation of the AT₂ receptor and will provide the basis for a rational approach to studies on the regulation of the AT₂ gene and its role in cell growth and possibly the cell cycle. On the basis of the present studies, we propose that the expression of the AT₂ gene is modulated in both positive and negative directions by different growth factors. To the best of our knowledge, this is the first report demonstrating that the AT₂ receptor and its mRNA are upregulated by growth factors such as insulin and IL-1ß and suppressed by bFGF, serum, or LPA.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1, AT2 = angiotensin II type-1 and -2 receptors bFGF = basic fibroblast growth factor C/EBP = CCAAT/enhancer binding protein FGF = fibroblast growth factor IL-1ß = interleukin-1ß IRS = insulin response sequence LPA = lysophosphatidic acid PDGF = platelet-derived growth factor PEPCK = phospho(enol)pyruvate carboxykinase PKC = protein kinase C Sarile = [Sar1,Ile8]Ang II TPA = tetradecanoylphorbol acetate

	Acknowledgments

 
This study was supported in part by research grants from the US Public Health Service (HL-14192 and HL-35323 from the National Institutes of Health). We thank Trinita Fitzgerald for excellent technical assistance in cell culturing.

Received April 17, 1995; accepted August 17, 1995.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Bottari SP, de Gasparo M, Steckelings UM, Levens NR. Angiotensin II receptor subtypes: characterization, signaling mechanisms, and possible physiological implications. Front Neuroendocrinol. 1993;14:123-171. [Medline] [Order article via Infotrieve]

2. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler RR, Saye JAM, Smith RD. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205-251. [Medline] [Order article via Infotrieve]

3. Sasaki K, Yamano Y, Bardhan S, Iwai N, Murray JJ, Hasegawa M, Matsuda Y, Inagami T. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type 1 receptor. Nature. 1991;351:230-233. [Medline] [Order article via Infotrieve]

4. Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233-236. [Medline] [Order article via Infotrieve]

5. Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T: Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993;268:24543-24546. [Abstract/Free Full Text]

6. Mukoyama M, Nakajima M, Horiuchi M, Sasamura H, Pratt RE, Dzau VJ. Expression cloning of type 2 angiotensin II receptor reveals a unique class of seven-transmembrane receptors. J Biol Chem. 1993;268:24539-24542. [Abstract/Free Full Text]

7. Ichiki T, Herold CL, Kambayashi Y, Bardhan S, Inagami T. Cloning of the cDNA and the genomic DNA of the mouse angiotensin II type 2 receptor. Biochim Biophys Acta. 1994;1189:247-250. [Medline] [Order article via Infotrieve]

8. Nakajima M, Mukoyama M, Pratt RE, Horiuchi M, Dzau VJ. Cloning of cDNA and analysis of the gene for mouse angiotensin II type 2 receptor. Biochem Biophys Res Commun. 1993;197:393-399. [Medline] [Order article via Infotrieve]

9. Tsuzuki S, Ichiki T, Nakakubo H, Kitami Y, Guo D-F, Shirai H, Inagami T. Molecular cloning and expression of the gene encoding human angiotensin II type 2 receptor. Biochem Biophys Res Commun. 1994;200:1449-1454. [Medline] [Order article via Infotrieve]

10. Webb ML, Liu EC-K, Cohen RB, Hedberg A, Bogosian EA, Monshizadegan H, Molloy C, Serafino R, Moreland S, Murphy TJ, Dickinson KEJ. Molecular characterization of angiotensin II type 2 receptors in rat pheochromocytoma cells. Peptides. 1992;13:499-508. [Medline] [Order article via Infotrieve]

11. Takahashi K, Bardhan S, Kambayashi Y, Shirai H, Inagami T. Protein tyrosine phosphatase inhibition by angiotensin II in rat pheochromocytoma cells through type 2 receptor, AT₂. Biochem Biophys Res Commun. 1994;198:60-66. [Medline] [Order article via Infotrieve]

12. Sumners C, Tang W, Zelezna B, Raizada MK. Angiotensin II receptor subtypes are coupled with distinct signal-transduction mechanisms in neurons and astrocytes from rat brain. Proc Natl Acad Sci U S A. 1991;88:7567-7571. [Abstract/Free Full Text]

13. Bottari SP, King IN, Reichlin S, Dahlstroem I, Lydon N, de Gasparo M. The angiotensin AT₂ receptor stimulates protein tyrosine phosphatase activity and mediates inhibition of particulate guanylase cyclase. Biochem Biophys Res Commun. 1992;183:206-211. [Medline] [Order article via Infotrieve]

14. Buisson B, Laflamme L, Bottari SP, de Gasparo M, Gallo-Payet N, Payet MD. A G-protein is involved in the angiotensin AT₂ receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells. J Biol Chem. 1995;270:1670-1674. [Abstract/Free Full Text]

15. Kang J, Posner P, Sumners C. Angiotensin II type 2 receptor stimulation of neuronal K⁺ currents involves an inhibitory GTP binding protein. Am J Physiol. 1994;267:C1389-C1397. [Abstract/Free Full Text]

16. Stoll M, Steckelings M, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT₂-receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.

17. Scheuer DA, Perrone MH. Angiotensin II type 2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am J Physiol. 1993;264:R917-R923. [Abstract/Free Full Text]

18. Cogan MG, Liu F-Y, Wong PC, Timmermans PBMWM. Comparison of inhibitory potency by nonpeptide angiotensin II receptor antagonists PD123177 and Dup 753 on proximal nephron and renal transport. J Pharmacol Exp Ther. 1991;259:687-691. [Abstract/Free Full Text]

19. Näveri L, Strömberg C, Saavedra JM. Angiotensin II AT₂ receptor stimulation increases cerebrovascular resistance during hemorrhagic hypotension in rats. Regul Pept. 1994;52:21-29. [Medline] [Order article via Infotrieve]

20. Grady EF, Sechi LA, Griffin CA, Schambelan M, Kalinyak JE. Expression of AT₂ receptors in the developing rat fetus. J Clin Invest. 1991;88:921-933.

21. Tsutsumi K, Saavedra JM. Characterization and development of angiotensin II receptor subtypes (AT₁ and AT₂) in rat brain. Am J Physiol. 1991;261:R209-R216. [Abstract/Free Full Text]

22. Tsutsumi K, Viswanathan M, Strömberg C, Saavedra JM. Type-1 and type-2 angiotensin II receptors in fetal rat brain. Eur J Pharmacol. 1991;198:89-92. [Medline] [Order article via Infotrieve]

23. Chiu AT, Herblin WF, McCall DE, Ardecky RJ, Carini DJ, Dunicia JV, Pease LJ, Wong PC, Wexler RR, Johnson AL, Timmermans PBMWM. Identification of angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;165:196-203. [Medline] [Order article via Infotrieve]

24. Sechi LA, Griffin CA, Grady EF, Kalinyak JE, Schambelan M. Characterization of angiotensin II receptor subtypes in rat heart. Circ Res. 1992;71:1482-1489. [Abstract/Free Full Text]

25. Suzuki J, Matsubara H, Urakami M, Inada M. Rat angiotensin II (type 1A) receptor mRNA regulation and subtype expression in myocardial growth and hypertrophy. Circ Res. 1993;73:439-447. [Abstract/Free Full Text]

26. Whitebread S, Mele M, Kamber B, de Gasparo M. Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Commun. 1989;163:284-291. [Medline] [Order article via Infotrieve]

27. Dudley DT, Hubbell SE, Summerfelt RM. Characterization of angiotensin II (AT₂) binding sites in R3T3 cells. Mol Pharmacol. 1991;40:360-367. [Abstract]

28. Dudley DT, Summerfelt RM. Regulated expression of angiotensin II (AT₂) binding sites in R3T3 cells. Regul Pept. 1993;44:199-206. [Medline] [Order article via Infotrieve]

29. Leung KH, Roscoe WA, Smith RD, Timmermans PBMWM, Chiu AT. Characterization of biochemical responses of angiotensin II (AT₂) binding sites in the rat pheochromocytoma PC12W cells. Eur J Pharmacol. 1992;227:63-70. [Medline] [Order article via Infotrieve]

30. Ichiki T, Inagami T. Transcriptional regulation of the mouse angiotensin II type 2 receptor gene. Hypertension. 1995;25(pt 2):720-725.

31. Angel P, Imagawa M, Chiu R, Stein B, Imbra RJ, Rahmsdorf HJ, Jonat C, Herrlich P, Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987;49:729-739. [Medline] [Order article via Infotrieve]

32. Chiu R, Boyle WJ, Meek J, Hunter T, Karin M. The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive gene. Cell. 1988;54:541-552. [Medline] [Order article via Infotrieve]

33. Akira S, Isshiki H, Nakajima T, Kinoshita S, Nishio Y, Hashimoto S, Natsuka S, Kishimoto T. A nuclear factor for the IL-6 gene (NF-IL6) interleukins: molecular biology and immunology. Chem Immunol. 1992;51:299-322. [Medline] [Order article via Infotrieve]

34. Isshiki H, Akira S, Tanabe O, Nakajima T, Shimamoto T, Hirano T, Kishimoto T. Constitutive and IL-1 inducible factors interact with the IL-1 responsive element in the IL-6 gene. Mol Cell Biol. 1990;10:2757-2764. [Abstract/Free Full Text]

35. Kambayashi Y, Bardhan S, Inagami T. Peptide growth factors markedly decrease the ligand binding of angiotensin II type 2 receptor in rat cultured vascular smooth muscle cells. Biochem Biophys Res Commun. 1993;194:478-482. [Medline] [Order article via Infotrieve]

36. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-PhOH-chloroform extraction. Anal Biochem. 1987;162:156-159. [Medline] [Order article via Infotrieve]

37. Moolenaar WH. Lysophosphatidic acid, a multifunctional phospholipid messenger. J Biol Chem. 1995;270:12949-12952. [Free Full Text]

38. Valius M, Kazlauskas A. Phospholipase C-1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993;73:321-334. [Medline] [Order article via Infotrieve]

39. Burgess WH, Dionne GA, Kaplow J, Mudd R, Friesel R, Zilberstein A, Schlessinger J, Jaye M. Characterization and cDNA cloning of phospholipase C-, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase. Mol Cell Biol. 1990;10:4770-4777. [Abstract/Free Full Text]

40. O'Brien RM, Granner DK. Regulation of gene expression by insulin. Biochem J. 1991;278:609-619.

41. Koike G, Horiuchi M, Yamada T, Szpirer C, Jacob HJ, Dzau VJ. Human type 2 angiotensin II receptor gene: cloned, mapped to the X-chromosome, and its mRNA is expressed in the human lung. Biochem Biophys Res Commun. 1994;203:1842-1850. [Medline] [Order article via Infotrieve]

42. Johnson PF, Landschulz WH, Graves BJ, McKnight SL. Identification of a rat liver nuclear protein that binds to the enhancer core element of three animal viruses. Genes Dev. 1987;1:133-146. [Abstract/Free Full Text]

43. Cao Z, Umek RM, McKnight SL. Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. Genes Dev. 1991;5:1538-1552. [Abstract/Free Full Text]

44. Poli V, Mancini FP, Cortese R. IL-6DBP, a nuclear protein involved in interleukin-6 signal transduction, defines a new family of leucine zipper proteins related to C/EBP. Cell. 1990;63:643-653. [Medline] [Order article via Infotrieve]

45. Hocke GM, Barry D, Fey GH. Synergistic action of interleukin-6 and glucocorticoid is mediated by the interleukin-6 response element of the rat 2 macroglobulin gene. Mol Cell Biol. 1992;12:2282-2292. [Abstract/Free Full Text]

46. Decombes P, Schibler U. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell. 1991;67:569-579. [Medline] [Order article via Infotrieve]

47. Williams SC, Cantwell CA, Johnson PF. A family of C/EBP-related proteins capable of forming covalently linked leucine zipper dimers in vitro. Genes Dev. 1991;5:1553-1567. [Abstract/Free Full Text]

48. Kinoshita S, Akira S, Kishimoto T. A member of the C/EBP family, NF-IL6ß, forms a heterodimer and transcriptionally synergizes with NF-IL6. Proc Natl Acad Sci U S A. 1992;89:1473-1476. [Abstract/Free Full Text]

49. Roman C, Platero JS, Shuman JD, Calame K. Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. Genes Dev. 1990;4:1404-1415. [Abstract/Free Full Text]

50. Ron D, Habener JF. CHOP, a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription. Genes Dev. 1992;6:439-453. [Abstract/Free Full Text]

51. Viswanathan M, Saavedra JM. Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing. Peptides. 1992;13:783-786. [Medline] [Order article via Infotrieve]

52. Dinarello CA. The biology of interleukin-1. Chem Immunol. 1992;51:1-32. [Medline] [Order article via Infotrieve]

53. Bondy CA, Werner H, Roberts CT Jr, LeRoith D. Cellular pattern of insulin-like growth factor-I (IGF-I) and type I IGF receptor gene expression in early organogenesis: comparison with IGF-II gene expression. Mol Endocrinol. 1990;4:1386-1398.[Abstract/Free Full Text]

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